1. Field of the Invention
Modern microbiological diagnostics and analysis serves for detection, enumeration, and identification of microorganisms present in different samples. In the areas of medical diagnostics and veterinary medicine, these organisms are pathogenic or dangerous microbes in human or animal blood, internal organs, skin, tissues, respiratory organs, and so on. In the area of industrial microbiology, microbes commonly pollute technological processes, materials, equipment, and finished products. In environmental analysis, there is often microbial contamination of water, indoor and outdoor air, and various surfaces. In epidemiology and biodefense—highly contagious pathogenic microorganisms from human body or environment.
Time, quality, and sensitivity of microbiological analyses are crucial for two reasons. First, tens of thousands of domestic microbiological laboratories spend several billions of dollars annually for products and processes quality control and prevention of contamination and spoilage in industry. These laboratories also spend money to provide diagnostic tests for humans, animals, plants, food, personal care products, soil, and environment. A quick, reliable, streamlined diagnostic test can save companies millions of dollars in the long run. Second, several thousands of people die even in such highly developed countries as the United States because of a delay in medical treatment caused by long-term diagnostics. Decreasing the time it takes to run these microbiological diagnostics tests will result in an increase of analysis reliability and sensitivity. Ultimately, this can save thousands of human lives worldwide.
This invention is a device for rapid detection and identification of microorganisms without preliminary growth.
2. Description of the Related Art
Modern methods of detection, enumeration, and identification of microorganisms can be divide into two main parts: 1) the methods and devices that need preliminary growth (enriching) to create a detectable amount of cells; 2) methods that don't need preliminary growth because they are capable of analyzing as little as a single cell.
The first group includes growth in solid or liquid regular or selective nutrient medias. It also includes several immunological methods. Examples are latex and hemagglutination, antibodies on magnetic particles, enzyme immunoassays like ELISA and Western Blot, and “deepstick” methods. The first group also includes chromatography of fatty acids, infrared Raman and FTIR spectroscopy, mass-spectrometry, and ATP-, bio-, and chemiluminescence. These methods require hundreds to millions of pure cells for detection of a certain microorganism, and, therefore, long (many hours or days) preliminary growth.
The second group of methods and devices does not need preliminary growth because they are capable of detecting and/or identifying even a single cell. These methods and devices consist of a group of nucleic acid methods like PCR and its various modifications, Epi-fluorescent methods (fluorogenic substrate methods, immunofluorescence), and a group of Flow Cytometry methods.
In addition to other drawbacks, methods of detection and/or identification of cells without preliminary growth usually need very expensive and sophisticated equipment and the work of high-level professionals. For example, devices used for PCR include the expensive thermocycler and sophisticated fluorometer. PCR is also used only for identification purposes. Enumeration of initial contamination is not reliable with PCR usage. PCR is very sensitive to organisms which can contaminate the test itself.
Epi-fluorescence usually needs only a fluorescent microscope to detect a single cell marked by a fluorescent dye or fluorescent antibody (Ab+fluorochrom). However, the amount of fluorescent substance present is restricted by volume of the cell body or cell surface. The small size of the object (single microorganism) makes the detection of one cell very difficult, especially with large background fluorescence usually present in a majority of the samples. Substances that flow out of a cell or enzyme immunoassay of a single cell are impossible with Epi-fluorescent methods because these indicator substances disperse in the surrounding space immediately but are not concentrated in a small volume like proposed by the current invention.
Flow Cytometry is based on a very complicated opto-electronics system. A flow cytometer consists of a sophisticated optical block, a block of electronics, a complicated hydrodynamic system, and a high-speed computer. Prices for different types of flow cytometers range from $50,000 to $140,000. Flow cytometers can analyze one single cell during its flow through channels with diameters of 10 microns each. This size of the channel is so narrow that it needs 17 hours to pass 100 ml of liquid through it, even if speed of flow is 20 meters per second. Therefore, flow cytometers are currently used in hematology because of large (5-6 million/ml) and more or less stable concentrations of blood cells. Also, flow cytometers very effective as sorters of cells mixtures in cytology. Usage of flow cytometry in microbiology is not easy. In microbiology these instruments are not often used because microorganisms can create clusters with other particles and can be confused with natural particles or dead cells. If the concentration of cells in a sample is very small, the time of analysis goes up dramatically. Therefore, preliminary concentration or even enriching is needed for Flow Cytometry microbiological applications. Microbes are also much more diverse by size and shape than blood cells and, thus, mistakes occur very often.
It is known, and currently used in practice, that dividing a sample into small volumes helps to detect cell concentration faster. This effect depends on reaching a detectable concentration in a small volume faster than in a large volume. U.S. Pat. No. 5,716,798 describes the method for rapid detection of microorganisms in a container divided on a plurality of discrete zones, each of which can be separately monitored for microbial presence by reaching detectable cell concentrations after preliminary growth in some zones. This method gives time-saving of 10% to 40% in comparison with other methods. U.S. Pat. No. 5,770,440 is based on the same effect. The present invention differs from these patents because of the analysis of a single cell. No time-consuming preliminary growth or nutrient media are necessary.
U.S. Pat. No. 4,959,301 is based on dividing a sample with viable biological entities into micro-droplets and detecting entities by growth or by biochemical reactions of a single entity within a droplet. This method can indicate a single cell in less than 30 minutes in some variants. Nevertheless, it is technologically complicated. Micro-droplets are produced with different volumes and require statistical analysis for calculating results. This method could be reproduced only in a laboratory by highly professional personnel with use of sophisticated and expensive equipment.
The proposed device has significant advantages in comparison with known methods:                It is capable of detecting and/or identifying by colored or fluorescent enzyme or enzyme-immunoassay as little as one single cell trapped in a micro-channel (one cell in one micro-channel corresponds to a concentration of 25 million cells per ml). Thus, no preliminary growth is needed, and a detectable concentration is reached in several minutes.        The price of the device and analysis is tens times less than Flow Cytometry or PCR (only a regular fluorescent or light microscope is needed for this diagnostic device). Also, the amount of reagent needed is substantially less than with the use of regular 96-well plates. As a result, the analysis is simple and cost-effective.        The device is simple to use and involves performing only regular filtration with just a few manipulations. Even non-specialists can easily adopt this device and procedure that is very important for their broad use.        Many different methods can be applied with the proposed device: detection of live cells by fluoregenic or chromogenic substrate(s), differentiation by special enzymes and artificial substrate(s) for them, identification by enzyme immunoassay of one single cell, analyzing of different liquid or air samples.        
These advantages promise excellent opportunities for implementation of this device and its versions in medical diagnostics, industry, environmental science, and biodefense.